Materials
Forty-eight male Sprague-Dawley (SD) rats (weighing 300±50 g) were provided by the Shanghai SLAC Laboratory Animal Co. Ltd. (license number: SCXK [Shanghai] 2012-0002). All animal experiments were performed in accordance with the relevant regulations of the Animal Protection Commission of Fujian province. D-gal was provided by AMRESCO. Interleukin (IL)-1, IL-6, and a tumor necrosis factor (TNF)-α lucume assay kit was provided by R&D.
Animals
The 48 rats were fed and provided water freely under standard conditions for 1 week. They were randomly divided into the following 4 groups: Model Groups A, B, C, and D (the control group). D-gal 1,000 mg·kg−1·d−1 was injected subcutaneously under the neck and back of each rat in the model groups for 1 week. Group D was subcutaneously injected with the same amount of normal saline for 1 week. The animal testing process complied with the relevant laws and regulations of the Animal Protection Commission.
The rats in each group were kept in cages in a clean animal laboratory in a quiet and well-ventilated environment with a temperature of 20–28 ℃, a relative humidity of approximately 50%, and 12 hours of alternate lighting. Four rats were kept in each cage and could feed and drink freely during the experiment period. Each rat was weighed once a day, and its general condition (i.e., hair, mental state, foraging behavior, and mobility) was observed.
Morris water maze (MWM) testing
The MWM test was performed 1 day before the injection of galactose and 24 hours after the injection. For the positioning and navigation experiment, the water pool was divided into 4 quadrants (I, II, III, and IV), and a quadrant was randomly selected as the starting quadrant. The rats were placed in a pool facing the pool wall, and the time between the rat’s entry into the water and when the rat found the platform (i.e., the escape latency period) was recorded. If the rat failed to find the platform after 90 s, it was guided to the platform and allowed to stay there for 15 s, and the escape latency period was recorded as 90 s. All 4 quadrants in the experiment had to be completed. The interval between the 2 experiments was 10–15 min. The experiment was repeated for 5 days. For the space exploration experiment, the escape platform was removed from the pool 1 day after the navigation experiment. The rats were placed in the water in the opposite quadrant of the original platform quadrant, and the time when the rats crossed the platform was recorded for up to 60 s.
Inhalation anesthesia experiment
After the completion of the experiment, each rat’s caudal veins were opened and connected to a trace pump. Next, the rat was placed in an acrylic, rectangle, transparent box with a 5-mm diameter hole on 3 sides. One of the side holes was connected to the oxygen flow pipe and another extension tube to monitor the concentration of sevoflurane in the box. The 2 other holes were used for the extension of the caudal vein and exhaust. Rats in Group A continuously inhaled 2% sevoflurane for 2 h, which was intraoperatively pumped with 0.25 mL·100 g−1·h−1 0.9% normal saline. Rats in Group B continuously inhaled 2% sevoflurane for 2 h, which was intraoperatively pumped with 10 μg·kg−1 dexmedetomidine. Rats in Group C were pumped normal saline (0.25 mL·100 g−1·h−1). Rats in Group D inhaled 2% sevoflurane for 2 h, which was intraoperatively pumped with 0.25 mL·100 g−1·h−1 0.9% normal saline. The Minimum Alveolar Concentration(MAC)values of sevoflurane and carbon dioxide at the end of the expiratory breath of the rats were monitored intraoperatively, and the breathing conditions of the rats were observed according to the respiratory waveform. The breathing rate, extremity color, and labial color of the rats were monitored during the operation.
Tissue preparation and histological assessment
The brain tissue (hippocampus) of the rats were harvested 12 hours after the MWM testing. The rats were anesthetized and perfused with normal saline, followed by 4% paraformaldehyde in a 0.1-m phosphate buffer (pH, 7.4). After perfusion, the brain tissue was quickly removed and fixed in a fixative solution for 24 h. The next day, paraffin embedding was performed, a coronal section of the hippocampal tissue was removed, and hematoxylin and eosin staining was performed. The pathological changes of the hippocampal tissues were observed under the light microscope under 100× and 400× magnification.
Enzyme-linked immunosorbent assay (ELISA) brain homogenate inflammatory mediator measurement
Twenty-four hours after the completion of the MWM testing, the rats were anesthetized with 10% chloral hydrate. The harvested brain tissue was quickly dried with filter paper, 4% phosphate-buffered saline homogenate medium was added, and the paper was homogenized in an ice bath. The concentration of IL-1, IL-6, and TNF-α in the homogenate was determined by an ELISA after the supernatant was extracted with a pipette for 20 min at 2,500 RPM.
Statistical analysis
Data are expressed as mean ± standard error of the mean. Data for rats’ weight, swim speed, platform crossing times, and inflammatory factor levels were tested for normality and variance homogeneity. When these conditions were met, group differences were evaluated by a 1-way analysis of variance (ANOVA), using the least significant difference for multiple pairwise comparisons. When the conditions of normality and variance homogeneity were not satisfied, group differences were evaluated using a nonparametric rank-sum test. Interactions between time (in the MWM testing procedure) and group factors were evaluated by a 2-way ANOVA with repeated measurements. P values less than 0.05 were considered statistically significant.